Real time imaging of single fluorophores on moving actin with an epifluorescence microscope

Sase, Ichiro; Miyata, Hidetake; Corrie, John E. T.; Craik, J. S.; Kinosita, Kazuhiko

doi:10.1016/s0006-3495(95)79937-4

Cited by 121 publications

(91 citation statements)

References 15 publications

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“…A 532-nm laser beam (DPSS 532-200, Coherent, Tokyo) was circularly polarized with a quarter-wave plate and introduced into a fluorescence microscope (IX70, Olympus, Tokyo) through a side port as described in ref. 4. To rotate the excitation polarization, a rotating sheet polarizer was inserted after the quarter-wave plate.…”

Section: Methodsmentioning

confidence: 99%

“…Individual behaviors can be assessed by single-fluorophore imaging (1)(2)(3)(4)(5), which is much less perturbing than imaging through a huge tag such as a plastic bead or actin filament (6). Real-time determination of fluorophore orientation (5,7,8) should be particularly useful, because a conformational change necessarily accompanies reorientation of one part against others.…”

mentioning

confidence: 99%

“…Measurement of rotation with a much smaller probe is desired. Thus, we examined rotational characteristics of F 1 with no load by attaching a single f luorophore to ␥ and assessing the f luorophore orientation through its polarized f luorescence on a microscope with an extremely low background (4,21). , and 50 pM Cy3-␣ 3 ␤ 3 ␥ in buffer A (50 mM KCl͞4 mM MgCl 2 ͞10 mM Mops-KOH, pH 7.0) was infused.…”

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confidence: 99%

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Stepping rotation of F ₁ -ATPase visualized through angle-resolved single-fluorophore imaging

Adachi

Yasuda

Noji

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

211

167

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Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F 1-ATPase revealed that the subunit rotates in the molecule in discrete 120°steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120°s tepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Stepping rotation of F ₁ -ATPase visualized through angle-resolved single-fluorophore imaging

Adachi

Yasuda

Noji

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

211

167

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show abstract

“…On the other hand, solution studies of actin and TMR-actin proved unsuitable to distinguish the open and closed structures. Earlier works using partially labeled TMR-actin had established that TMR-actin copolymerizes with unlabeled actin (14,15), thus enabling monitoring of actin dynamics in live cells by microinjecting trace amounts of TMR-actin (16). Recent work further showed that unpolymerizable TMR-actin causes destabilization of standard actin filaments upon copolymerization (17,18).…”

mentioning

confidence: 99%

Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with Actin Regulatory Proteins

Conchaudron

Didry

et al. 2006

Journal of Biological Chemistry

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The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP-or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys374 by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5-adenylyl ␤,␥-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP-or ADP-bound state.The regulated dynamics of actin assembly plays a pivotal role in cell motility. The hydrolysis of actin-bound ATP that accompanies actin polymerization is at the origin of treadmilling, which powers actin-based motility processes. Hydrolysis of ATP destabilizes actin-actin interactions in the filament and affects the structure of actin enough to specify the recognition of ATP-and ADP-actin by different regulatory proteins, yet the nature of the structural change that supports the functional importance of this reaction in actin following ATP hydrolysis remains a debated issue.Crystallographic data indicate that the nucleotide-binding cleft of actin can be in an open or closed state; however, the open and closed states do not appear to logically correlate with the bound nucleotide being ADP (1-3) or ATP (4 -9). Reconstructions of actin filaments indicate that the structure of ADP-F-actin is more consistent with the open state of actin (10, 11). The crystal structure of actin in complex with ciboulot, a Wiskott-Aldrich syndrome homology 2 (WH2) 2 domain/␤-thymosin repeat-containing protein that, like ␤-thymosin, specifically binds ATP-actin, shows that it is only in the closed state of actin that the conformation of the shear zone of actin between subdomains 1 and 3 allows the binding of WH2 domains (3). These two studies (3, 10) suggest that ATP-actin has a closed conformation...

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“…These include e.g., a modified version of flow cytometry [63,64 ] used for single fluorophore detection and nearfield microscopy [65][66][67], evanescent-field fluorescence microscopy (EFFM, also called total internal reflection microscopy [68]) [69], and versions of epi-fluorescence microscopy [70][71][72][73] used for single fluorophore imaging. In the case of low temperature experiments, reviewed in Ref.…”

Section: Introductionmentioning

confidence: 99%

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Zheng¹,

Goldner²,

Leuba³

2007

Methods

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Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/ acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

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Real time imaging of single fluorophores on moving actin with an epifluorescence microscope

Cited by 121 publications

References 15 publications

Stepping rotation of F ₁ -ATPase visualized through angle-resolved single-fluorophore imaging

Stepping rotation of F ₁ -ATPase visualized through angle-resolved single-fluorophore imaging

Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with Actin Regulatory Proteins

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

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Real time imaging of single fluorophores on moving actin with an epifluorescence microscope

Cited by 121 publications

References 15 publications

Stepping rotation of F 1 -ATPase visualized through angle-resolved single-fluorophore imaging

Stepping rotation of F 1 -ATPase visualized through angle-resolved single-fluorophore imaging

Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with Actin Regulatory Proteins

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

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Product

Resources

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Stepping rotation of F ₁ -ATPase visualized through angle-resolved single-fluorophore imaging

Stepping rotation of F ₁ -ATPase visualized through angle-resolved single-fluorophore imaging